Journal of Chromatography A, 658 (1994) H-53

Elsevier Science B.V., Amsterdam

CHROMSYMP. 2905

Review

Chromatographic procedures for phytoecdysteroids

R. Lafont
Departement de Biologie, Laboratoire de Biochimie, CNRS URA 686, kcole Nonnale Suptrieure, 46 Rue d’Ulm,
75230 Paris Cedex 05 (France)

E.D. Morgan* and I.D. Wilson

Department of Chemistry, Keele University, Keele, Staffordshire, ST5 SBG (UK)

ABSTRACT

The complexity of the mixtures of ecdysteroids in plants and the close similarities in their chemical structures have challenged
chemists to find suitable ways to separate and identify them. Great ingenuity has been applied to these problems and
consequently a wide range of separation and methods are available today. These methods have been reviewed with assessment of
their strengths and limitations, with the intention to guide investigators towards the methods most useful to their purpose.

CONTENTS

1. Introduction ............................................. .. 32
2. Sample preparation ........................................ .. .. .. .. .. 32
2.1. General considerations ................................. .. .. .. .. .. .. .. .. . .. 32
2.2. Partition techniques .................................... .. .. .. .. .. . .. .. .. 32
2.2.1. Solvent partitioning .............................. .. .. .. 32
2.2.2. Liquid-liquid partition techniques ................... .. .. 33
2.2.2.1. Counter-current distribution ................. .. .. 33
2.2.2.2. Droplet counter-current chromatography ....... .. .. . .. .. .. .. 34
2.3. Low-pressure column chromatography ..................... .. .. .. .. .. .. .. .. .. .. 34
2.3.1. Analytical scale: disposable cartridges for sample clean-up .. .. .. .. .. .. .. 34
2.3.1.1. Normal-phase cartridges .................... .. 34
2.3.1.2. Reversed-phase cartridges .................. .. .. 34
2.3.1.3. Immobilized phenylboronic acid (PBA) cartridges .. .. .. .. .. .. 35
2.4. Preparative scale ...................................... .. .. .. .. .. .. .. .. .. 35
2.5. Selected examples of protocols ........................... .. .. .. .. .. . .. .. .. 37
3. High-performance liquid chromatography ...................... .. .. .. .. .. .. .. 37
3.1. Correspondence between TLC and HPLC .................. .. .. 37
3.2. Chromatographic procedures ............................ .. .. .. 38
3.2.1. Normal-phase systems ............................ .. .. .. .. 38

* Corresponding author.

0021-9673/94/$26.00 (Q 1994 Elsevier Science B.V. All rights reserved

SSDZ 0021-9673(93)E0838-L
32 R. Lafont et al. I 1. Chromatogr. A 658 (1994) 31-53

3.2.2. Reversed-phase systems ......................... .. .. 40

4. Planar chromatography ................................... .. .. 40
5. Gas chromatography ..................................... .. .. .. 43
6. Supercritical fluid chromatography .......................... .. .. 45
7. Capillary electrophoresis .................................. .. .. 47
8. Hyphenated techniques of chromatography and mass spectrometry . .. 50
9. Future prospects ........................................ .. .. 51
References ............................................... .. 51

1. INTRODUCTION and low-pressure column chromatography of

The short period between 1965 and 1967 was
one of remarkable advances in the study of the
insect moulting hormones or ecdysteroids. In
1965, eleven years after their initial isolation of
the compound, Karlson et al. [l], using an
improved isolation procedure, obtained sufficient
crystalline ecdysone from silkworm pupae for
Huber and Hoppe to determine that it was a
polyhydroxysterol [2]. The next year Hampshire
and Horn [3] showed their crustecdysone, iso-
lated from a crayfish, Jasus lulundii was 20-
hydroxyecdysone and Hocks and Wiechert [4]
isolated 20-hydroxyecdysone from the silkworm
Bombyx mori, but more important for our pres-
ent subject, the data becoming available made
others realize that compounds they were examin-
ing from plants had the same or similar struc-
tures. Very quickly Nakanishi et al. [5] reported
ponasterone A from the leaves of the coniferous
tree Podocarpus nakai, and Galbraith and Horn
[6] isolated 20-hydroxyecdysone from the Aus-
tralian pine Podocurpus elutus. In the following
year Takemoto et al. [7] isolated 20-hydroxy-
ecdysone and inokosterone from the common
Japanese weed Achyranthes fauriei and Jizba et
al. [8] reported the isolation of 20-hydroxy-
ecdysone from the roots of the fern Polypodium
w&are.
Thus began the remarkable series of inves-
tigations of plants for ecdysteroids, which still
continues today. Galbraith and Horn [6] sug-
gested in their paper that the ecdysteroids in
plants, or phytoecdysteroids may protect plants
from insect attack. Thus, too began the con-
troversy, still unsettled, over the function, if any,
of ecdysteroids in plants.
The isolations referred to above were all
carried out using solvent extractions, partitions
various kinds, with only a little help from thin
layer chromatography. The best of these meth-
ods are listed here. The most efficient methods
of separation and the most sensitive methods of
detection were developed subsequently, largely
stimulated by the search through insect and plant
materials for ecdysteroids.
2. SAMPLE PREPARATION
2.1. General considerations
The ecdysteroids form a group of rather polar
compounds and as a consequence the initial
extraction step is best performed using a polar
solvent such as methanol (MeOH). Alternative
solvents include ethanol (EtOH), acetone, ace-
tonitrile and methanol-water mixtures. Super-
critical fluid extraction would also be adequate,
but it was not used up to now in this case.
Having obtained and concentrated an extract,
the next step usually involves one or more
solvent partitions with the aim of removing the
bulk of polar and non-polar contaminants prior
to chromatography (Fig. 1).
2.2. Partition techniques
2.2.1. Solvent partitioning
In the early studies [9], the initial partition was
made between an aqueous concentrate and IZ-
butanol (BuOH). The BuOH residue, into which
the ecdysteroids were extracted, was then par-
titioned between aqueous MeOH and hexane to
remove non-polar material such as lipids. How-
ever, reversing this order of operation was found
to be beneficial and led to reduced emulsion
formation during the partition step and fewer
problems with frothing during evaporation.
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53 33

TABLE 1
PARTITION COEFFICIENTS OF ECDYSTEROIDS AND
VARIOUS PRECURSORS

K = Concentration in the non-polar phase/concentration in

the polar phase.
cr&&mll-Wuq(l:l) : l3oAc-wata :

I ~umol~Wata(P.lNNaOIi.~rAfOH)
~maJl&water(l:l:l)
1 1~ tplum>Mculfald
water (5:4:1); Ropmool-~w~....

Ecdysteroid K Ref.

Cyclohexane-n-butanol-water (5:5:10)
Ecdysone 3.54 71
Makisterone A 1.27 72
20-Hydroxyecdysone 0.52 71
3-Epi-20-hydroxyecdysone 0.52 72
26-Hydroxyecdysone 0.39 73
20,26_Dihydroxyecdysone 0.06 72
n-Butanol-water (19)
I Ecdysone cu. 10 74
FINAL PURIFICATION OR ANALYSIS 20-Hydroxyecdysone 5.3 9
~in-kyp.~&b+wWliquiddmnufography
Ethyl acetate-water (1:l)
2,22-Dideoxyecdysone 20 116
Fig. 1. General extraction and purilication chart for ecdy-
2-Deoxyecdysone 4
steroids.
Ecdysone 0.4
20-Hydroxyecdysone 0.1
Chloroform-methanol-water (2:l:l)
Other suitable partitioning systems for removing 2,22-Dideoxyecdysone 13 116
lipids include hexane-aqueous methanol (7:3, ZDeoxyecdysone 2.7
v/v) or light petroleum (b.p. 4040°C)-aqueous Ecdysone 0.4
20-Hydroxyecdysone 0.1
methanol. Mixtures of water-propanol (PrOH)
(3:1, v/v) and hexane can also be used to Chloroform-ethanol-water (1:l:l)
remove non-polar contaminants, the ecdyster- Ecdysone 4.6 116
20-Hydroxyecdysone 1.5
oids remaining in the aqueous phase. The addi-
tion of (NH,)$O, to promote the formation of Chloroform-water (1:l)
2,22,25Trideoxyecdysone (Ketodiol) 90 116
the two phases may be required with the PrOH
2,22_Dideoxyecdysone 20
mixture. The separation of polar impurities from 2-Deoxyecdysone 2.9
the ecdysteroids can be achieved by partition Ecdysone 0.06
between water and BuOH (ecdysteroids partition 20-Hydroxyecdysone 0.015
into the organic phase) and water and ethyl Hexane-acetonitrile (1:l)
acetate (EtOAc) (ecdysteroids remain in the Cholesterol 1.5 116
aqueous phase). 2,22,25-Trideoxyecdysone 0.06
The major factors governing the choice of 2,22_Dideoxyecdysone co.01
2-Deoxyecdysone co.01
solvent partition system are the type of con- Ecdysone co.01
taminants to be removed (i.e. mainly lipids or
mainly polar, etc.) and the nature of the ecdy-
steroids to be isolated. Thus the addition or
removal of one OH group, or conjugation to
polar (e.g. sulphate) or non-polar (e.g. acetate or
fatty acyl) groups can significantly affect parti- 2.2.2. Liquid-liquid partition techniques
tion ratios. Partition coefficients of representa- 2.2.2.1. Counter-current distribution (CCD).
tive ecdysteroids and precursors in a number of Counter-current distribution between BuOH and
systems are given in Table 1. water is effective for removing polar contami-
34
nants (the addition of a small amount of salt
reduces emulsion formation) and CHCl,-
MeOH-water or hexane-PrOH-water.
This technique, as is the case for all other
liquid-liquid partitions systems, has the advan-
tage of a 100% sample recovery. The technique
is not of wide use at the present time. It has been
advantageously replaced by the more recent
DCCC and RLCC techniques (see below).
2.2.2.2. Droplet counter-current chromatog-
raphy (DCCC). This technique provides an effi-
cient means for purifying samples up to the gram
range. It belongs to the family of liquid-liquid
partition chromatographic methods.
DCCC equipment is compact, it can be used
with rather crude samples, and in favourable
cases it can allow the preparation of “pure”
compounds [lo-131. In our hands, it seemed
however to require a subsequent HPLC step in
order to get fully pure ecdysteroids. Several
systems in the ascending or descending mode
have been described (Table 2).
Rotation locular counter-current chromatog-
raphy (RLCC) was designed as an alternative to
DCCC [14]. It was applied to the purification of
Vitex strickeri methanolic extracts [15], in combi-
nation with recycling HPLC.
Although efficient, DCCC and RLCCC are
rather time-consuming, and processing a single
sample usually requires l-5 days [14]. This is
because of the need to allow good exchange to
proceed between the mobile droplets and the
stationary phase. To overcome this drawback,
HSCCC (high-speed counter-current chromatog-
TABLE 2
SOLVENT SYSTEMS FOR DROPLET COUNTER-CURRENT
A = Ascending mode; D = descending mode.
Solvent system
CHCl,-MeOH-water (13:7:4)
CHCl,-C,H,-EtOAc-MeOH-water (45:2:3:60:40)
C,H,-CHCl,-MeOH-water (5:5:7:2)
CHCI,-MeOH-water (65:20:20)
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53
raphy) was proposed [14]. In that case, the
column is a multi-layer coil and efficient mixing
is achieved through planetary rotation. Centrifu-
gal force replaces normal gravity and allows
separation to be achieved in hours rather than
days.
2.3. Low-pressure column chromatography
2.3.1. Analytical scale: disposable cartridges for
sample clean-up
2.3.1.1. Normal-phase cartridges. The use of
low pressure chromatography on a small column
has been used very early in phytoecdysteroid
research for the fractionation of crude extracts.
It was at that time either silica or alumina which
was used in such columns (normal phase sys-
tems), generally eluted with binary mixtures -a
step-gradient of alcohol in chloroform or ben-
zene. Use of these disposable cartridges is now
generally called solid-phase extraction (SPE).
2.3.1.2. Reversed-phase cartridges. The availa-
bility of hydrophobic phases (resins like Amber-
lite or hydrocarbon-bonded silica) had led to a
complete renewal of the procedures [16]. More-
over, the design of small cartridges or syringes
containing 0.2-l g of HPLC phase has led to an
ideally suited material for a rapid clean-up of
small samples.
Among them, Sep-Pak cartridges from Waters
are the most widely used (Fig. 2 [17-211. In fact
their use has been extended not only for bio-
logical extracts but also for desalting purposes,
e.g. direct adsorption of ecdysteroids from cul-
CHROMATOGRAPHY
Mode Ref.
A 10, 11, 13,50,7.5
D 23
D 24
D 24, 50
R. Lafont et al. I 1. Chromatogr. A 658 (1994) 31-53
I OR
5 ml mthtutol-warn 13 5 ml ItEthmol
1 Q
I specific developments.
s ml mthanol-water 31
Fig. 2. Utilization of reversed-phase cartridges
steroid purification.
ture media or from reverse phase HPLC frac-
tions containing an involatile buffer.
The use of C,, cartridges may be extended to
more refined separations, provided that rigorous
protocols are used regarding solvent composition
and volume. By that way, it appears possible to
separate ecdysteroids that differ by the presence
or absence of a single -OH group (provided that
its position on the molecule evokes a significant
polarity change).
2.3.1.3. Immobilized phenylboronic acid
(PBA) cartridges. Recently, cartridges filled with
immobilized phenylboronic acid on silica, aga-
rose or cellulose have become available. Such
systems are able to retain selectively some com-
pounds bearing vicinal diols. Ecdysteroids may
contain such diol functions, i.e. at C2-C3, C20-
C22 and/or C25-C26. Up to now, experiments
have been made with compounds bearing no
26-OH, and therefore were limited to the two
first cases [22].
It appears that the 2,3-diol does not readily
form cyclic boronates (at least with a 2-OH,, and
3s
a 3-OH,,), and the adsorption process is essen-
tially determined by the presence or absence of
the 20,22-diol, e.g. it is particularly efficient to
distinguish between ecdysone and 20-hydroxy-
ecdysone series. It is thus possible to adsorb
ecdysteroids and to elute them sequentially, first
ecdysone-type and then 20-hydroxyecdysone-
type compounds (Fig. 3).
I This procedure can be combined with other
solid phase systems to separate various classes of
ecdysteroids [22] and it could have several
2.4. Preparative scale
Low-pressure column chromatography uses
either normal phases or (less often) reversed
phases (Table 3). The size of the column has to
be related to the size of samples (at least ten
times the dry weight of the sample), and elution
is usually performed with a step-gradient. Most
for ecdy-
often, silica or alumina are used with a step-
gradient of methanol or ethanol in a chlorinated
solvent (chloroform or methylene chloride). In
the reversed-phase mode, Sephadex LH-20 may
be used to remove non-polar contaminants, and
polyamide is used specifically to remove yellow
pigments.
70% u&n in 34blmicacid
70% ueol-l in
alkahbuffu acidic buffer in7O%MeOH
Fig. 3. Extraction procedure for ecdysteroids using immobil-
ized phenylboronic acid cartridges.
36 R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53

TABLE 3
USE OF LOW-PRESSURE CHROMATOGRAPHY FOR MEDIUM- OR LARGE-SCALE PURIFICATION

Type of stationary phase Solvent system Ref.

Normal phases
Silica (silica gel, silicic acid CHCI,-MeOH (step gradient) 76
or celite) CHCl,-MeOH (step gradient) 77
CHCl,-MeOH (step gradient) 78
CHCl,-MeOH (step gradient) 79
(+ 1% CH,COOH)
CHCl,-MeOH (8:2) 80
CHCl,-MeOH (955) 81
CHCl,-MeOH (100:3) 82
CHCl,-EtOH (19:l) 83
Me&O-CH,Cl,-water (2:8:1), 84
then EtOH
CHCl,-MeOH-water (82~1 or 85
7.5:2:1, lower phase)
CH,Cl,-EtOH (step gradient) 49
EtOAc 86
EtOAc-MeOH (step gradient) 87
Alumina CHCl,-MeOH (step gradient) 77
(containing usually 10% water) CHCl,-MeOH (step gradient) 78
CHCl,-MeOH (step gradient) 88
CHCl,-MeOH (step gradient) 89
CHCl,-MeOH (2:l) 90
CHCl,-EtOH (step gradient) 91
CH,Cl,-EtOH (step gradient) 92, 93
EtOAc-EtOH (step gradient) 94
Me,CO-CH,Cl,-water 84
(62.5:15:10)
CH,CI,-EtOH (9:l) 95
EtOAc-MeOH (1:l) 76
EtOAc-EtOH (1:l) 83
EtOAc-EtOH (2:l) 96
Sephadex LH-20 CH,Cl,-Me,CO 83, 97
CH,Cl,-MeOH (step gradient) 83, 97
CHCl,-EtOH (88:12) 96
Reversed-phases”
Amberlite XAD-2 Water-MeOH (step gradient) 98,99
(medium-pressure LC)
Sephadex LH-20 EtOH-water (7:3) 81
MeOH 85, 92, 93
Celite impregnated with Water (saturated with BuOH 76
BuOH + cyclohexane + cyclohexane)
Polyamide Water 80, 93, 100
(for removal of flavonoids)

’ RP is used in a broad sense, to include less conventional systems.

 37
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53

Podocw n&Iii leaves

cm&!)
I
Extract with 80% EtOH
canalnnu

1
33% Me-OH 70% MeOH (3 I) 100% MeOH (4 I)
Polarimpvitis Ponaaaonc A (60 mg)
Traces of ecdystcroids
(Ponasterme c) I
chmmatography on a silica column

I
Ponasterone A (6.1 g)
Ponasterone B (0.05 g)
Ponasuronc c (O.Sg)

Fig. 4. Isolation of ecdysteroids from Podocarpus nakaii leaves [16].

2.5. Selected examples of protocols provide unambiguous evidence for the identity of
a given compound.
Some representative protocols are given in When facing a separation problem it is pos-
Figs. 4-7, that each use a part of the above sible to make a logical choice, according to the
techniques. Other complex processing schemes results of a preliminary TLC step. Current
can be found elsewhere [23-251. HPLC techniques have been designed about ten
years ago and the HPLC of ecdysteroids may
now be considered to be a mature technique (for
3. HIGH PERFORMANCE LIQUID reviews see refs. 26-31).
CHROMATOGRAPHY

3.1. Correspondence between TLC and HPLC

HPLC is the most popular technique for
ecdysteroid separations, both for analytical and
It is known that relationships between TLC
preparative purposes. It offers wide choice of
and HPLC may help to design adequate HPLC
techniques, that are adapted for polar or non-
solvent systems. The migration of a given com-
polar metabolites. The identification of any ecdy-
pound by TLC is given by its R, (=retardation
steroid by co-migration with a reference com-
factor). Another parameter also used for TLC is
pound must rely on the simultaneous use of
the R,, defined as
several (at least two) different HPLC systems,
generally one normal-phase and one reversed- R, = log( 1 /RF - 1)
phase system. Given the very large ecdysteroid
family, there is no guarantee that these criteria Any change on a molecule evokes a change of
38 R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53

Chippeddried wood (13 kg)

I
Fmly v-
ground
Exawted with petmlcum edw.r(->discaided)
Extrxt with EtOH I
Exuactcd with Chlomfotm-MeOH-Ammonia (9:0.9%1)
I Ftutbcr extracted with Chlomform
concennatc
I
0.25 M NaOH 4-w n-Butanol Combined exlmcts concentrated t0 l/10
I StMdfoK44da~atOT
BaCkW&Cd n-Butanol~~, 0.1 M NaOH

O.OzH cl--e n-Butanol ‘Ad Mother-liqum

I I Exaaacd intowarn
concenasred
B&&&d
DiscaIded
COtlCi”Wi@ Dried

I I
Methanol-WY 1:l *L, Chl~fOIm solids
Combined
Triturat~Iwithwater(-> insotubleresiduel)
Chlomfotm _I_ Methanol-water B* I
I
canccn~
Ttiturafed with water (--> insoluble tesiduc2)
Chmmatography on silicic acid
I
I
chromatography on c&e MSS
cIystalliscdinanicebatb
I I
Rccrysrallise
fromEtoAc-water
1O:l
I
2044YDROXYECDYSON!Z(170 mg)

Fig. 5. Isolation of 20-hydroxyecdysone from Podocarpus

bark [115]. h4&ZWWZA Motherliquors chromatography on silica
(Cww EVaporared
I I

the R, or R, and the same change on two 20-HydmxyeCdySMe 20-Hydroxyecdysone

molecules of the same family may evoke the Fig. 6. Isolation of ecdysteroids from Kalaaizna seeds [lOBI.
same AR,. Accordingly, the AR, values are
cumulative, and two independent changes 1 and AR, = log k; - log ki = log(kalki), or
2 evoke a final AR, = AR,, + AR,,.
When using HPLC, the behaviour of a given AR, = log (Y
compound is expressed by its retention time (or
The consequence of this is that a given modi-
volume) t, or better by its capacity factor k’,
fication (e.g. presence or absence of a given
defined as k’ = (rR - t,lt,), where t, is the re-
-OH group) evokes a similar effect on various
tention time of a compound eluted in the void
compound pairs; for instance the (Yvalue for the
volume. The correspondence with TLC is easy to
ecdysone:20-hydroxyecdysone pair is the same
make, as
as for the 2-deoxyecdysone:2-deoxy-20-hydroxy-
f, = t,lR,, k’ = (l/R, - 1) and thus R, = log k’ ecdysone one, provided of course that isocratic
conditions are used. Moreover, it seems possible
Therefore, provided that the chromatographic
to determine the expected retention time of a
conditions and the phases are the same, it is easy
compound where no reference is available [32].
to deduce the HPLC behaviour from the TLC
Similar calculations apply to acetates, conju-
data, and the relationship between R, and k’ is
gates, etc. and may prove useful in many cases.
exemplified in Fig. 8. It is clearly apparent from
it that HPLC solvents might be chosen from
among those giving R, between 0.1 and 0.3, in 3.2. Chromatographic procedures (Table 4)
order to avoid a too rapid elution or extended
analysis times. 3.2.1. Normal-phase systems
Moreover, the selectivity factor LXfor a pair of Normal-phase (NP) systems generally use sil-
compounds 1 and 2 analyzed by HPLC can be ica columns (sometimes aminopropyl- or diol-
defined as CY= kilki, and from this bonded columns) and a standard system of
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53 39

Dliedmaedsample TABLE 4

I CHROMATOGRAPHIC SYSTEMS COMMONLY USED

Bxtmaedwithmethanol:Bxuact&iedlnvacuunl; FOR THE HPLC ANALYSIS OF ECDYSTEROIDS
R&due panitlened z rnz 80% MeOH-water
See also ref. 26 for a more extensive review of data before
I 1980.
Methanolphase
I
Concenm in vacuum and subjected to reversed-phase Mode of chromatography Ref.
flash chromatography log/lOOgFS’silica gel. eludng
200 ml fractions, water to MeOH in 10% steps
Normal-phase chromatography (silica or diol-,
I aminopropyl or TMS-bonded silica)
Ekdysteroid 6wxions
Chloroform-95% aqueous ethanol 101
I Chloroform-ethanol (gradient) 96
Cuncentcatedin vacuum and subjected to normal phase Chloroform-isopropanol (gradient) 81
flash chromatogmphy3g13Ogsilica gel, eludng
30 ml fractions, chloroform to MeOH in 10% steps Chloroform-methanol 79
Dichloromethane-tetrahydrofuran-methanol 42
I Dichloromethane-methanol 42
Ecdysteroidfractions
Dichloromethane-ethanol-water 33
I Dichloromethane-isopropanol-water 33
Concentratedin vacuum and’~bjected ~_~m&o~~~y
on LH20 Se hadex using D&lomme Dichloromethane-methanol-water-acetic acid 103
Dr‘ch!oromethane-MeOHgradientelution Dichloroethane-methanol-isopropanol
Hexane-ethanol-methanol-acetonitrile 104
I
Isooctane-isopropanol-water 105
Isooctane-isopropanol-water (with grad.
methanol) 38
Fig. 7. Isolation of ecdysteroids from plants (after Russel Cyclohexane-isopropanol-water 32
and Greenwood [97]). Reversed-phase chromatography [Amberlite XAD-2
or (mainly) C,, bonded silica]
dichloromethane-isopropanol-water mixtures as Ethanol-water 98
initially designed by Lafont et al. [33]. The Methanol-water 16
Methanol-water 106
respective proportions of the three components
Methanol-water 39
can vary, according to sample polarity. Thus, Methanol-water 85
specific ternary mixtures can be prepared for Acetonitrile-water 107
non-polar compounds, e.g. acetates or ecdysone Acetonitrile-water 81
precursors (e.g. 125:15:1, v/v/v) for medium- Acetonitrile-water 39
Acetonitrile-methanol-water 75
polarity compounds (125:25:2 or 125:30:2 for
Acetonitrile-0.1% TFA 75
ecdysone and 20-hydroxyecdysone) or for more Dioxan-water 39
polar ecdysteroids (125:40:3 for 26-hydroxy- Tetrahydrofuran-water 39
ecdysteroids; 100:40:3 for glucosides). Methanol-triethylammoniurn phosphate +
With normal-phase systems, k’ is particularly 1-butanesulfonic acid 95
Isopropanol-water, 50°C 108
Recycling HPLC (Asahipack GS-320)
Methanol 15, 40

24

sensitive to temperature. Low temperatures may

10 result in greatly decreased retention times, pos-
sibly because they reduce ecdysteroid solubility
in the stationary water phase adsorbed onto the
0
silica particles. This effect is particularly striking
0.00 0.20 0.40 0.60 0.80 1.00 between 10°C and 20°C. As a consequence, it is
RF highly recommended to place the HPLC system
Fig. 8. Inverse relationship between k’ (HPLC) and R, in a room where temperature is controlled.
VW. Polar-bonded columns (diol or aminopropyl

silica) can also be used instead of silica ones
[27,34]. Diol-bonded columns have been used
with Chenopodium album and Kochia scoparia
(Chenopodiaceae) extracts [35,36] whereas
aminopropyl ones proved particularly efficient
for the separation of mixtures of 3a-OH, 3P-OH
and 3-oxoecdysteroids [37] -indeed these sepa-
rations were better than those achieved on silica
columns [3]. In addition, bonded phases allow
the use of gradients without the problems linked
with long re-equilibration times encountered
with silica columns.
Non-polar bonded-phase columns can also be
used with the above solvents, and they provide
somewhat efficient separations with reduced re-
tention times [38]. In this respect, trimethylsilane
(TMS) bonded phases seem particularly interest-
ing: they give very symmetrical peaks and a
selectivity that differs from silica columns. The
retention of ecdysteroids on non-polar bonded
columns results from the presence of remaining
free silanol groups on bonded phases (this be-
comes especially evident when columns have
previously been used over a long period) which
could in theory permit both NP- and RP-HPLC
with a single column! There exist some HPLC
columns (cyanopropyl or nitrile-bonded) that
have been designed both for NP and RP pur-
poses and these might probably be used for
normal-phase chromatography of polar (but of
course non-ionic) ecdysteroids.
Dichloromethane-based solvents, although
very efficient for chromatographic separations,
suffer from a high UV-cutoff and quenching
properties of this compound, which preclude the
use of diode-array detectors or in-line radioac-
tivity monitors. This problem may be overcome
with isooctane-based mixtures [29] which in
counterpart suffer from a lower efficiency (plate
number) and poor ecdysteroid solubility, which
may become inconvenient for (1) polar ecdy-
steroids and (2) preparative purposes. The selec-
tivity of such solvent systems is also very differ-
ent from that of dichloromethane-based ones.
More recently, [32] cyclohexane-based mixtures
were tested: they provide much better solubili-
ties and allow the use of diode-array detectors.
On the other hand, these mixtures display a
significantly higher viscosity and hence working
R. Lafont et al. 1 J. Chromatogr. A 658 (1994) 31-53
pressure, a problem that can be overcome by
increasing solvent temperature.
3.2.2. Reversed-phase systems
Reversed-phase HPLC with C,,-bonded col-
umns is the most widely used system, and it
provides efficient separations. Methanol-water
mixtures are usual, and they represent the
easiest way to obtain satisfactory separations,
even if they are not the most efficient ones.
Other water-miscible organic solvents may
equally be used, and acetonitrile provides the
advantage of forming mixtures with water that
have a much lower viscosity. On the other hand,
50% methanol is a much better solvent than
20-25% acetonitrile for preparative purposes.
Selectivity linked with the nature of the or-
ganic phase is an important parameter for de-
termining the most adequate solvent mixture for
a given separation: depending upon the specific
problem, better results may be obtained either
with methanol, acetonitrile or THF [32,39].
Recycling HPLC using methanol as eluent
may provide an interesting method for the res-
olution of closely migrating compounds and it
was successfully used for the separation of ecdy-
steroids from the bark of Vitex strickeri [15]. This
method appears to have several advantages,
regarding its ability to separate closely related
compounds at the preparative scale and its low
solvent consumption [40].
4. PLANAR CHROMATOGRAPHY
Planar chromatography, particularly thin-layer
chromatography (TLC) has been used extensive-
ly for the qualitative analysis and isolation
of phytoecdysteroids. Paper chromatography,
whilst now generally considered to be obsolete,
has been used for the separation of ecdysteroids
[41] and for completeness these results are pre-
sented in Table 5.
NP-TLC systems using silica as stationary
phase have most frequently been used to sepa-
rate ecdysteroids. A widely used general solvent
system has been chloroform-ethanol (4:1, v/v)
with a single ascending development, however,
many normal phase solvent systems have been
described for these and details of a number of
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53 41

TABLE 5
R, OF ECDYSTEROIDS ON PAPER CHROMATOGRAPHY [41]

Solvent systems are prepared by saturating the non-polar component (A) with the mixture (B + C) of polar solvents.

Compound Solvent system Ratio B:C R,

A B C

Ecdysone Benzene-propan-2-ol-water 5545 0.69

Toluene-propan-2-ol-water 50:50 0.34
Toluene-butan-2-ol-water 50:50 0.79
Water-butan-l-01 Saturated” 0.68
20-Hydroxyecdysone Benzene-propan-2-ol-water 55:45 0.34
Benzene-butan-2-ol-water 55:45 0.29
Toluene-butan-2-ol-water 50:50 0.45
Toluene-butan-2-ol-water 6040 0.29
Toluene-propan-2-ol-water 50:50 0.12
Water-butan-l-01 Saturated’ 0.80
Butan-1-ol-water Saturated” 0.86
20-Hydroxyecdysone Toluene-propan-2-ol-water 50:50 0.46
2-acetate
Inokosterone Benzene-propan-2-ol-water 55:45 0.45
Benzene-butan-2-ol-water 55:45 0.38
Toluene-butan-Zol-water 50:50 0.53
Toluene-butan-2-ol-water 6040 0.37
Toluene-propan-2-ol-water 50:50 0.15
Water-butan-l-01 Saturated” 0.74
Butan-1-ol-water Saturated” 0.88
Makisterone A Benzene-propan-2-ol-water 55:45 0.52
Toluene-propan-2-ol-water 50:50 0.22
Toluene-butan-2-ol-water 50:50 0.64
Ponasterone A Benzene-propan-2-ol-water 55:45 0.93
Toluene-propan-2-ol-water 50:50 0.74
Toluene-butan-2-ol-water 50:50 0.92

a First liquid saturated with the second.

solvent systems used to separate the ecdysteroids
are given in Table 6 [42]. The R, values obtained
for some 20 compounds in the chloroform-etha-
nol (4:l) solvent system are provided in Table 7
[43]. Following separation by NPTLC the detec-
tion of ecdysteroids on TLC plates can be
accomplished in a number of ways. Where the
plates incorporate a suitable fluorescence in-
dicator that can provide a general, if rather
non-specific method of detection. Spray reagents
have been used to provide a higher degree of
specificity, and the vanillin-sulphuric acid re-
agent has been particularly widely employed.
This reagent gives a range of colour with differ-
ent ecdysteroids as indicated in Table 8. The
colours produced range from pink (e.g. cyas-
terone) and red (e.g. 20-hydroxyecdysone) to
dark green (e.g. ecdysone), but like many such
colour reactions are subject to quite wide inter-
laboratory variations. Table 8 also lists a number
of additional chromogenic reactions which have
been utilised for these compounds. Scanning
densitometry now provides a convenient method
for obtaining in situ UV spectra, and this can
provide increased confidence in the identification
of sample components as ecdysteroids (e.g. see
42 R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53

TABLE 6

SOLVENT SYSTEMS FOR TLC OF ECDYSTEROIDS ON SILICA GEL

Data from ref. 42.

Solvent system Composition R,

Ecdysone 20-Hydroxyecdysone

CHCl,-95% aqueous EtOH 7:3 0.39 0.34

CHCl,-95% aqueous EtOH 13:7 - 0.31
CHCl,-EtOH 3~2 - 0.5
CHCl,-MeOH 9:l 0.10 0.07
CHCl,-MeOH 5:l - 0.23
CHCl,-MeOH 3:2 - 0.40
CHCl,-MeOH 1:l - 0.55
CHCl,-MeOH-25% aqueous NH,-water 12:7:1:1 - -
CHCl,-MeOH-AcOH 4:1:0.05 - 0.36
CHCl,-propan-l-01 9:5 0.21 0.12
CHCl,-MeOH-Me,CO 6:2:1 - 0.48
CHCl,-MeOH-Me,CO 6:2:1 - 0.33
CH,Cl,-Me,CO-MeOH 2:l:l 0.69 0.62
CH,Cl,-Me,CO-EtOH 16:4:5 0.32 0.10
CH,Cl,-MeOH-C,H, 25:5:3 - 0.19
CH,Cl,-Me,CO-water 15:62.5:10 0.65 _
CH,Cl,-MeOH-water 7.9:15.1 0.32 0.19
CH,Cl,-MeOH-25% aqueous NH,-water 77:20:2:1 0.47 0.40
CH,Cl,-MeOH 7:3 - -
C,H,-MeOH 7:3 0.40 _
EtOAc-EtOH 4:l _ 0.60
EtOAc-EtOH 4:l 0.49 0.46
EtOAc-EtOH 4:l - 0.32
EtOAc-EtOH-water 2:8:1 - -
EtOAc-MeOH-NH,-water 10:2:1:1 - -

Fig. 9). As discussed in more detail later, in situ
mass spectrometry has also been used to good
effect for the identification of phytoecdysteroids
following NP-TLC.
RP-TLC chromatography on bonded phases
(C,, C,, C12, C1*, aminopropyl and cyano-
propyl) as well as on paraffin-impregnated silica
gel (e.g. refs. 39, 44 and 45) has also been
employed for ecdysteroids and typical results,
obtained using various C,, bonded phases, for a
range of structures are provided in Table 9 [45].
In general, methanol-water (l:l, v/v) solvent
systems provide good separations but other or-
ganic modifiers can be used to achieve different
selectivities [44]. Reversed-phase systems, like
their HPLC equivalents are poor at separating
5P-OH compounds from their 5/3-H analogues
(e.g. polypodine B from 20-hydroxyecdysone).
The effects of various substitution patterns on
the RP-TLC of ecdysteroids are discussed in
detail elsewhere [45]. Detection methods follow-
ing RP-TLC are essentially the same as those
employed for NP-TLC separations, although RP-
TLC-MS has not proved to be so readily
achieved as NP-TLC-MS .
Separations can also be modified, ‘in both NP-
and RP-TLC systems by esterifying 20,22-diol-
containing ecdysteroids with boronic acids (e.g.
phenylboronic acid) 146,471. This has the effect
of reducing the polarity of such esters with a
consequent effect on the chromatographic sepa-
ration of an ecdysteroid mixture. This can be
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53 43

TABLE 7

R, AND ReedysoneVALUES OBTAINED FOR ECDYSTEROIDS BY NP-TLC ON SILICA GEL TLC PLATES USING
CHLOROFORM-ETHANOL (4:l)

Data from ref. 43.

Compound R, R ecdysone Compound R, R ecdyrons

Poststerone 0.32 152 22Isoecdysone 0.13 62

2-Deoxyecdysone 0.38 180 Calonysterone 0.42 200
Ecdysone 0.21 100 Pterosterone 0.32 152
20-Hydroxyecdysone 0.15 71 Kaladasterone 0.49 233
Muristerone A 0.27 129 Pinnasterol 0.56 267
Dacrysterone 0.27 129 20-Hydroxyecdysone 2-cinnamate 0.53 252
2-Deoxy-20-hydroxyecdysone 0.31 141 Polypodine B 2-cinnamate 0.56 267
Makisterone A 0.20 95 Acetylpinnasterol 0.68 324
Polypodine B 0.22 104 Ponasterone C 0.38 181
Inokosterone 0.17 77 Ponasterone C 2-cinnamate 0.65 310
2-Deoxy-3-epiecdysone 0.44 209 Ponasterone A 0.42 200
Ajugasterone C 0.22 104 Carpesterol 0.86 410
Cyasterone 0.33 157

used to good effect to resolve ponasterone A and
2-deoxyecdysone, which are otherwise difficult to
separate [4,6].
Of the more recently developed techniques for
planar chromatography, automated multiple de-
velopment (AMD) and over-pressure layer chro-
matography (OPLC) have been applied to the
resolution of ecdysteroids [48,49]. AMD pro-
vided good separations but was somewhat time-
consuming [48]. The use of OPLC for the sepa-
ration of phytoecdysteroids of Silene nutans [49]
enabled very rapid analysis to be performed, and
it could easily be used for preparative work.
However, the time required for plate prepara-
tion, etc., means that this is unlikely to supplant
conventional separation methods.
The current practice of TLC for the analysis of
phytoecdysteroids is to use it for qualitative
applications such as monitoring extractions and
purification (see refs. 50 and 51 for some recent
examples). In these applications the ease of TLC
and the generally rugged nature of the separa-
tion system are well suited to a rapid and high
throughput of often difficult samples. However,
there is no practical reason why scanning den-
sitometry could not be used for quantitative
analysis of plant extracts expecially with the high
concentrations of material frequently encoun-
tered in such samples. It therefore seems likely
that, with the wider availability of scanning
densitometers that the use of TLC and HPTLC
for quantitative analysis of these compounds will
increase.
5. GAS CHROMATOGRAPHY
Ecdysteroids are too polar and have too little
thermal stability to be suitable subjects for gas
chromatography (GC) but by protecting some or
all of the hydroxyl groups as silyl ethers they can
be thermally stabilized and their polarity reduced
so that they can be chromatographed in the
gaseous phase. The gas chromatography of
ecdysone as a trimethylsilyl ether derivative was
first described by Katz and Lensky [52], although
the exact nature of the product was not stated.
Morgan and Woodbridge [53,54] developed con-
ditions for the preparation of trimethylstyl ether
0-methyloximes for the quantitative study of
ecdysteroids, but later it was found that protec-
tion of the ketone group as an 0-methyloxime
was not necessary. Poole et al. [55] and Borst
44 R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53

TABLE 8
REACTIONS OF ECDYSTEROIDS AND THEIR DERIVATIVES WITH SPRAY REAGENTS

Compound Reagent Colour Ref.

Cyasterone Vanillin-sulphuric acid Pink 9

Dacrysterone Vanillin-sulphuric acid Mauve-brown 109
2-Deoxy-20- Vanillin-sulphuric acid Olive green then 9
hydroxyecdysone yellow
Ecdysone Vanillin-sulphuric acid Blue then red 9
brown
Vanillin-sulphuric acid Turquoise 9
Vanillin-sulphuric acid Red-brown 110
2&Dinitropheny Slightly yellow 110
hydraxine, then
R,Fe(CN),
Phosphotungstic acid Blue 110
20,26_Dihydroxyecdysone Vanillin-sulphuric acid Turquoise 111
20-Hydroxyecdysone Vanillin-sulphuric acid Olive green then 9
brown
Vanillin-sulphuric acid Yellow-green 9
Vanillin-sulphuric acid Turquoise-grey 110
blue
2,4-Dinitrophenyl Slightly yellow
hydraxine, then
&Fe(CN),
Phosphotungstic acid Blue
20-Hydroxyecdysone Vanillin-sulphuric acid Olive green 112
2-cinnamate
Inokosterone Vanillin-sulphuric acid Orange 9
Makisterone A Vanillin-sulphuric acid Mauve-brown 9
Vanillin-sulphuric acid Mauve-brown 109
Makisterone C Vanillin-sulphuric acid Dark green 9
Vanillin-sulphuric acid Green 109
Ponasterone A Vanillin-sulphuric acid Mauve then grey- 9
green
Ponasterone B Vanillin-sulphuric acid Blue-mauve then 9
brown
Ponasterone C Vanillin-sulphuric acid Green then brown 9
Vanillin-sulphuric acid Dark green 112
Ponasterone C 2- Vanillin-sulphuric acid Dark green 112
cinnamate
Polypodine B 2-cinnamate Vanillin-sulphuric acid Olive green 112
Pterosterone Vanillin-sulphuric acid Dark green 112
Green 9
Rubrosterone Vanillin-sulphuric acid Brown 9
20-Hydroxyecdysone Vanillin-sulphuric acid Olive green then 113
2-acetate yellow
20-Hydroxyecdysone Vanillin-sulphuric acid Olive green then 113
3-acetate yellow
20-Hydroxyecdysone Vanillin-sulphuric acid Dark green 113
22-acetate
20-Hydroxyecdysone Vanillin-sulphuric acid Olive green then 113
2,3-diacetate yellow
20-Hydroxyecdysone Vanillin-sulphuric acid Dark green 113
2,22-diacetate
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53 45

TABLE 8 (continued)

Compound Reagent Colour Ref.

20-Hydroxyecdysone Vanillin-sulphuric acid Dark green 113

3,22diacetate
20-Hydroxyecdysone Vanillin-sulphuric acid Dark green 113
2,3,22&acetate
20-Hydroxyecdysone Vanillin-sulphuric acid Dark green 113
2,3,22,25_tetracetate
2tSHydroxyecdysone Vanillin-sulphuric acid Olive green then 113
20,22-acetonide brown
20-Hydroxyecdysone Vanillin-sulphuric acid Olive green then 113
2,3,20,22-diacetonide brown

derivative formation have replaced GC we are

I. 1. I‘ I1 I 1
20 60
mm4’
Fig. 9. HP-TLC of an extract from the plant Silene schafta,
silica gel, chloroform-ethanol (4:1, v/v), 254 nm. Peaks:
1 = 20-hydroxyecdysone; 2 = polypodine B.
and O’Connor [56] almost simultaneously discov-
ered that ecydsone trimethylsilyl ethers were
suitable subjects for the electron capture detec-
tor which made the sensitivity and selectivity of
detection of ecdysteroids much greater. This
work was done with packed columns, and was
reviewed in 1976 [42], but the difficulties of
preparing trimethylsilyl ethers in a reproducible
and quantitative manner [57] have discouraged
the use of gas chromatography in ecdysteroid
work.
The introduction of fused-silica capillary col-
umns increased the resolution of GC enormously
so that the resolving power of GC and the
sensitivity of electron-capture detection have yet
to be bettered by any physical methods for
ecdysteroids. Because other chromatographic
methods described here which do not require
not aware of the use of GC in any phytoecdy-
steroid work in the past decade.
An example of capillary column operating
conditions for trimethylsilylated ecdysteroids is
given by Evershed and co-workers [58,59]. They
used a 25 m x 0.22 mm fused-silica capillary
column, coated with 0.1 pm film of BP-l (a
non-polar dimethylsiloxane polymer) with oven
temperature 50°C at injection, then raised imme-
00 diately to 200°C and programmed at 8°C min-’
to 320°C and held at that temperature. Using
helium as the moving phase at a linear velocity
of 100 cm s-l, they found ecdysone trimethylsilyl
ether had a retention time of 15.2 min, 20-
hydroxyecdysone 16.0 min, makisterone A 17.2
min and 20,26-dihydroxyecdysone 18.2 min [58].
6. SUPERCRITICAL FLUID CHROMATOGRAPHY
Separation of complex mixtures can be
achieved by chromatography using a moving
liquid or gaseous phase. It was pointed out by
Lovelock in 1958 (see ref. 60) that a supercritical
fluid should also act as a mobile phase for
chromatography. The supercritical fluid used in
most cases is carbon dioxide or carbon dioxide
“modified by” (i.e. mixed with) methanol, or
some other small-molecule polar substance to
increase the polarity of the supercritical fluid.
The instrumentation is either an adaptation of
GC, using fused-silica capillary columns and a
flame ionization detector or an adaptation of
HPLC, using packed columns and a UV detec-
46 R. Lafont et al. I J. Chromatogr. A 6.58 (1994) 31-53

TABLE 9

RETENTION OF PHYTOECDYSTEROIDS ON RP-TLC PLATES OF DIFFERENT ORIGINS

Solvent system: methanol-water (5050, v/v). Data from ref. 45.

Compound Merck C,, bonded TLC Whatman C,, bonded Macherey-Nagel C,,
plates TLC plates bonded TLC plates

hR, %R, ecdysone hR, %R, ecdysone hR, %R, ecdysone

Ecdysone 29 100.0 28 100.0 32 100.0

20-Hydroxyecdysone 2-cinnamate 4 13.8 3 10.7 5 15.6
Inokosterone 44 151.8 37 132.0 45 140.6
20-Hydroxyecdysone 44 151.8 38 135.7 49 153.1
Muristerone A 32 110.3 31 110.7 45 140.6
Carpesterol 0 0.0 0 0.0 0 0.0
2-Deoxy-20-hydroxyecdysone 21 72.4 29 103.6 29 90.6
Ponasterone C 2-cinnamate 0 0.0 0 0.0 0 0.0
Pterosterone 29 100.0 38 135.7 37 115.6
Abutasterone 42 144.8 56 200.0 52 162.5
Integristerone A 45 155.2 63 225.0 52 162.5
Calyonysterone 20 68.9 37 132.1 24 75.0
Kaladasterone 17 58.6 30 107.1 30 93.8
Ponasterone A 16 55.2 18 64.3 24 75.0
Makisterone A 31 106.9 40 142.9 40 125.0
Dacrysterone 31 106.9 37 132.1 40 125.0
Polypodine B 42 144.8 44 157.1 49 153.1
Cyasterone 40 137.9 51 182.1 49 153.1
Acetylpinnasterol 0 0.0 0 0.0 0 0.0
2-Deoxyecdysone 1.5 51.7 17 60.7 17 53.1
Ajugasterone C 38 131.0 38 135.7 44 137.5
Ponasterone C 29 100.0 37 132.1 40 125.0
Poststerone 37 127.6 38 135.7 46 143.8

tor. The flame detector precludes the use of
organic modifiers but has great sensitivity and
resolution. Raynor et al. [61] have used capillary
supercritical fluid chromatography (SFC) to
chromatograph some relatively non-polar ecdy-
steroid precursors but the retention times of
ecdysone and hydroxyecdysone were too great
for practical purposes. Perhaps these experi-
ments would be worth repeating using higher
pressures and possibly slightly higher tempera-
ture. Now that the electron capture detector is
becoming adaptable to SFC conditions, it be-
comes more important to attempt capillary SFC
of ecdysteroids. SFC with electron-capture de-
tection could become a superior method for
ecdysteroids in terms of resolution, sensitivity
and selectivity. Morgan et al. [62] have shown
that packed-column SFC using carbon dioxide-
methanol mixtures is a practical procedure, both
for pure ecdysteroids and for plant extracts (see
Fig. 10) [63]. Very short columns can be used
with advantages of short retention times and
high resolution. The sensitivity of detection is
greater than with HPLC probably as a result of
the sharper peaks and greater UV transparency
of the fluid [64]. Packed-column SFC has the
retention characteristics of a NP-HPLC system.
A variety of columns from silica to ODS can be
used. In practice, aminopropyl silica and cyano-
propyl silica seem best for ecdysteroids. Some
conditions are given in Table 10. A further
advantage of SFC over HPLC is the avoidance of
costly high purity solvents and the problems of
their recovery and disposal. Since many ecdy-
steroids contain vicinal diols, particularly 2,3-
and 20,22-diols, reagents specific for vicinal diols
R. Lafont et al. I J. Chromatogr. A 6.58 (1994) 31-53 47

A 0 4 used for the separation of phytoecysteroids.

MCE was performed using fused-silica polyim-
ide-coated capillaries of 50 or 75 pm I.D. and 72
or 50 cm in length, respectively. The run buffer
4 employed was 40 mM sodium dihydrogen
2 phosphate-20 mM disodium borate-20 mM so-

!LL
1 dium dodecyl sulphate-methanol
7
612345 012345
Min
Fig. 10. Supercritical fluid chromatograms of plant extracts.
(A) Silene otites and (B) Silene nutans [63]. Peaks: 1 = 2-
deoxyecdysone; 2 = 2-deoxy-20-hydroxyecdysone; 3 = poly-
podine B; 4 = 20-hydroxyecdysone; 5 = 26-hydroxypoly-
podine B; 6 = integristerone A; 7 = 20,26dihydroxyecdy-
sone. Conditions: column 5 pm cyanopropyl silica (20 x 4.6
mm) CO,-MeOH (9:l) at 3 ml min-‘, 60°C and 290 bar,
detection at 235 nm.
can be useful in selecting out such compounds
from mixtures. We have described the use of
solid-phase extraction with immobilized boronic
acids for the selective retention of ecdysteroids
containing 20,22-diols [22]. We have recently
extended this to the preparation and separation
by SFC of methyl, butyl and phenylboronic
esters of ecdysteroids with 20,22-diol groups
[651.
7. CAPILLARY ELECTROPHORESIS
Capillary zone electrophoresis (CZE) has
been shown to provide very efficient separations
for a wide range of substances. As the bulk of
the ecdysteroids are uncharged, simple CE,
which relies on the presence of an ionised group
to attain separations, cannot be used. However,
micellar capillary electrophoresis (MCE) , where-
by a molecule such as sodium dodecyl sulphate is
added to the buffer at a concentration above its
critical micelle concentration, has been shown to
be suitable for such compounds. In a series of
preliminary studies we have evaluated MCE
[66,67] for a range of sample types, including
plant extracts of various degrees of purity. These
studies clearly demonstrated that MCE could be
(5%, v/v) at
pH 9.4. Depending upon the system, separations
were performed at 20 or 8.5 kV and a run
temperature of 50°C. Increasing either the ap-
plied voltage or temperature reduced migration
times. The organic modifier content of the mo-
bile phase also affected the migration of the
ecdysteroids with elution time increasing with
organic modifier content.
The migration times of a number of common
phytoecdysteroids, in the two CZE systems ex-
amined, with the run buffer described above are
given in Table 11. Using a 50 pm I.D. capillary,
migration times ranged from 7.2 min for cyas-
terone up to 25.9 min for 2-deoxyecdysone. The
observed elution order is similar to that seen in
RP-HPLC. This is not surprising as in MCE the
separation mechanism for ecdysteroids is based
on hydrophobic partitioning. It is interesting to
note that there is an excellent separation be-
tween ponasterone A (16.5 min) and 2-deoxy-
ecdysone (25.9 min) which have been difficult to
separate by RP-HPLC, TLC, etc. The results
obtained with a 75 pm capillary were similar.
MCE gave a linear calibration curve over the
range 0 to 560 pg/ml, corresponding to 0 to 2.8
ng injected on-column. The sensitivity of the
instrument was such that the detection of cu. 175
pg (35 pg/ml) on-column of a sample of pure
ecdysone was possible without difficulty. Ecdy-
steroid-rich extracts from both plant and insect
sources were subjected to analysis by MCE.
Samples with a range of purities were examined
with different degrees of success. Attempted
MCE of simple methanol extracts of Silene
nutuns were unsuccessful because large and dis-
torted peaks were obtained, apparently due to
column overloading and the presence of interfer-
ing compounds. The analysis of plant extracts
after more extensive purification gave good re-
sults as illustrated in Fig. 11 for an extract of
Silene o&es. This shows the sample to contain
20-hydroxyecdysone , 2-deoxy-20-hydroxyecdy-
48 R. Lafont et al. I 3. Chromatogr. A 658 (1994) 31-53

TABLE 10
EXAMPLES OF CONDITIONS USED FOR SUPERCRITICAL FLUID CHROMATOGRAPHY OF ECDYSTEROIDS

1 p.s.i. = 6894.76 Pa. For many other conditions, see ref. 114.

Column type Column Mobile phase Flow-rate Pressure or Temperature Compound ra Ref.
dimensions density (“C) @in)

Cllpilltl~
Cyanopropyl- 10mxS pm CO, - 0.4-0.71 g 120 14,22,25-Tridwxyecdysone 20 61
dimethylsiloxane (1:l) cK3 2-Deoxyecdysone 31 61

Packed
Cyanopropyl 250 x 4.6 mm CO,-MeOH 3.25 I min-’ 300 bar 50 14,22,25_Trideoxyecdysone 1 61
Spherisorb 5 pm (9O:lO) gas 2-Deoxyecdysone 2.3 61
Ponasterone A 2.5 61
Ecdysone 3.2 61
Polypodine B 3.2 61
Makisterone A 3.2 61
20-Hydroxyecdysone 4.0 61
Cyasterone 7.0 61

HypersiI5 pm 100 x 4.6 mm CO,-MeOH 4 ml min-’ 300 bar 80 Ecdysone 1.68 62

(8Oz20) 20-Hydroxyecdysone 1.88 62
Inokosterone 2.10 62
Cyasterone 1.71 62

Cyanopropyl Spherisorb 250 x 46 mm CO,-MeOH 3 ml min-’ 290 bar 60 2-Deoxyecdysone 1.5 63

(9O:lO) 2-Deoxy-20-hydroxy-
ecdysone 1.6 63
Polypodine B 1.8 63
20-Hydroxyecdysone 2.0 63
%Hydroxypolypodine B 2.2 63
Integristerone A 2.4 63
20,26_Dihydroxyecdysone 2.6 63

Silica 5 pm 250 x 4.6 CO,-MeOH 3.0 2000 p.s.i. 60 Ecdysone 3.0 64

(7525) 20-Hydroxyecdysone 3.2 64
(80:20) 2.0 2OC0p.s.i. 60 Ecdysone 7.1 64
20-Hydroxyecdysone 8.1 64

ODS 5 pm 250 x 4.6 CO,-MeOH 3.0 2OLXlp.s.i. 60 Ecdysone 2.3 64

(75:25) 20-Hydroxyecdysone 2.3 64
(86:14) 3.0 2QOOp.s.i. 60 Ecdysone 4.3 64
20-Hydroxyecdysone 4.7 64

Aminopropyl 250 x 4.6 CO,-MeOH 2.0 2NlO p.s.i. 60 Ecdysone 4.5 64

(80:20) 20-Hydroxyeulysone 4.8 64
Cyanopropyl 250 x 4.6 CO,-MeOH 2.0 2000 p.s.i. 80 Ecdysone 5.2 64
(8020) 20-Hydroxyecdysone 6.0 64
Aminopropyl 250 x 4.6 CO,-MeOH 2.0 2000 p.s.i. 40 Ecdysone 11.0 64
(80~20) 20-Hydroxyecdysone 13.9 64

sone and 2-deoxyecdysone as major components. hydroxyecdysone , polypodine B and 2-deoxy-20-

The electropherogram obtained for the analysis hydroxyecdysone . Under these conditions 20-hy-
of an extract of the roots of Slene nutuns is droxyecdysone and polypodine B co-migrate
shown in Fig. 12. This extract contained 20- (Fig. 12a). This lack of separation between
R. Lafonr et al. I 1. Chromatogr. A 658 (1994) 31-53 49

TABLE 11 polypodine B and 20-hydroxyecdysone is similar

RETENTION TIME DATA FOR ECDYSTEROIDS FOR to that found in RP-HPLC. However, the res-
MICELLAR CAPILLARY ELECTROPHORESIS olution of these compounds is possible by MCE
providing that run buffers containing over 50%
MCE conditions as in text, data from ref. 67. A = ABI 27OA (v/v) of methanol are used, albeit with long
CZE system; B = Beckman P/ACE 2000 CZE system.
analysis times (over 30 min). Increasing the
applied voltage did enable analysis times to be
Compound Retention time reduced without loss of resolution as shown in
(min) Fig. 12b.
MCE therefore is capable of providing effi-
A B
cient separations of phytoecdysteroids in plant
Calonysterone 14.43 - extracts. On-column sensitivity is excellent, and
Cyasterone 7.17 - clearly adequate for these concentrated samples.
2-Deoxyecdysone 25.91 21.74 MCE can provide an alternative to HPLC or
2-Deoxy-20-hydroxyecdysone 12.84 11.90
TLC in this type of application and the small
Ecdysone 11.30 10.65
7.35 7.34
on-column sample requirements may also prove
20-Hydroxyecdysone
20-Hydroxyecdysone Zcinnamate 13.57 - useful. However, relatively pure extracts seem to
Kaladasterone 13.19 - be required for good peak shape. Sensitivity is
- 7.34
Polypodiie B limited by the amount of material (a few
Ponasterone A 16.45 16.02
nanolitres) that can be introduced into the capil-
Pterosterone 10.07 -
8.89 9.08 lary. The relatively low concentration, as op-
Makisterone A
Muristerone 8.37 - posed to on-column sensitivity of MCE, may
therefore be a source of difficulty for some

1
a b

Od-

0 ;
0

10 20 30
Timelmin
Fig. 11. MCE of an ecdysteroid-rich extract of the plant
Silene otites using the ABI 270A. The extract contained, in
order of elution, (1) 20-hydroxyecdysone, (2) 2-deoxy-ZO-
hydroxyecdysone and (3) 2-deoxyecdysone. Conditions: UV
absorption at 240 nm, mobile phase 5% methanol and 95%
buffer containing 40 mM NaH,PO,, 20 mM Na,HBO, and
20 mM sodium dodecyl sulphate, pH 9.4, 20 kV
Timelmin
Fig. 12. MCE, using the Beckman P/ACE, of an ecdy-
steroid-rich extract of the plant Silene nutans using (a)
standard conditions in which polypodine B (1) and ZO-
hydroxyecdysone (2) co-migrate, but are resolved from 2-
deoxy-20-hydroxyecdysone (3); and (b) 52.5% of methanol
in the run buffer when 20-hydroxyecdysone elutes before
polypodine B. Conditions as in Fig. 11, but varying the
mobile phase in (b) and using 8.5 kV throughout.
50
sample types. Whilst these initial studies are
promising it is too early to say whether or not
MCE will be generally useful for the analysis of
phytoecdysteroids. Further work is required to
enable a rigorous comparison with other ana-
lytical techniques to be made.
8. HYPHENATED TECHNIQUES OF
CHROMATOGRAPHY AND MASS SPECTROMETRY
The large number of phytoecdysteroids that
have been encountered in plant extracts clearly
poses problems in their identification based
merely on chromatographic properties alone.
Whilst selectivity can be attained by the use of a
number of different chromatographic systems
(see also Lafont et al. [32]), linked chroma-
tography-mass spectroscopy provides an alter-
native system for unequivocally establishing
identity. The use of GC-MS for ecdysteroids is
well established for insect and other arthropod
samples but has not been widely applied to plant
samples. The reason for this is that in the case of
arthropod samples it is the sensitivity as well of
the specificity of the mass spectrometer as a
detector which is the driving force for its use. In
the case of plant samples the quantities of
material are usually such that it is considered to
be easier to isolate material for mass spec-
trometry rather than go through the process of
derivatisation which is necessary before GC can
be used. The introduction of SFC has also
enabled the ecdysteroids to be introduced into
the mass spectrometer without the need for
derivatisation, and SFC-MS has been used for
the identification of a number of ecdysteroids in
extracts of Silene nutans and Silene otites [63].
The results obtained were either chemical ioniza-
tion- or electron-impact-like, depending upon
the temperature of the jet block, with higher
temperatures associated with greater fragmenta-
tion. Conditions could thus be varied to give
molecular mass or diagnostic fragmentation data.
In the case of HPLC-MS for phytoecdyster-
oids progress has been limited by the available
interfaces. In Fig. 13 the chromatograms ob-
tained for HPLC-MS of an extract of Silene
otites with UV and MS data, monitoring the ions
R. Lafont et al. / J. Chromatogr. A 6.58 (1994) 31-53
1ooa uv 240 nm
1 A
10
SCAN No.
Fig. 13. HPLC-MS of ecdysteroids present in an extract of
Silene otites separated by RP-HPLC on a C,, bonded column
(ODS Spherisorb 20 x 0.46 cm) using acetonitrile-0.1 M
ammonium acetate (4060) at 1 ml min-’ with a thermospray
interface (VG Quatro, capillary temperature 270°C). (a) UV
trace at 240 nm; (b) ion current for m/z 481 for 20-hydroxy-
ecdysone; (c) ion current m/z 465 for 2-deoxy-2-hydroxy-
ecdysone and (d) ion current m/t 449 for 2-deoxyecdysone.
at m/z 481, 465 and 449 are shown using the
thermospray interface. The resulting spectrum
for 20-hydroxyecdysone (m/z 481) is given in
Fig. 14A. It is typical of the results obtained with
this interface following reversed-phase HPLC
(methanol-ammonium acetate buffer, see cap-
tion for details). A number of ions are associated
with the 20-hydroxyecdysone peak corre-
sponding to the protonated molecular (MH) ion
(481), MH - 18 (463) and MH -2 x 18 (445)
(loss of one or two molecules of water) and an
ion corresponding to M + MeCN + NH: (539),
highlighting the difficulties of interpretation en-
countered with this mode of ionisation. In order
to characterise more fully such compounds with
this interface we have recently employed MS-
MS techniques. An example of the results ob-
tained is shown in Fig. 14B where the daughter
ion spectrum obtained for the ion at m/z 481 is
shown. The weak ion at m/z 463 (not marked)
and ions at m/z 445 and 427 correspond to loss
of 1, 2 and 3H,O respectively, but no simple
explanation can be given for the other ions.
Similar spectra were obtained for 2-deoxy-20-
hydroxyecdysone and 2-deoxyecdysone.
In addition to the hyphenation of SFC and
R. Lafont et al. I J. Chromatogr. A 658 (1994) 31-53
Fig. 14. (A) The mass spectrum obtained for 20-hydroxy-
ecdysone using the conditions outlined in Fig. 13. (B) The
daughter ion spectrum obtained for the M + 1 ion (m/z 481)
for 20-hydroxyecdysone.
HPLC with MS and MS-MS techniques we have
also experienced considerable success in combin-
ing TLC separations with fast atom bombard-
ment MS [68] and MS-MS [69,70], using both
“in-line” and “off-line” techniques. The inherent
simplicity of this approach (especially the off-line
technique) makes it an attractive and readily
implemented alternative to the method of hyphe-
nation outlined above. Furthermore, as the TLC
plate effectively stores the separation, confirma-
tion of identity by MS need not be performed at
the time of separation (or indeed at the same
site).
9. FUTURE PROSPECTS
It would appear that all the stratagems and
inventions of the chromatographer have been
applied to the separation of phytoecdysteroids
from their matrix of plant substances. Tech-
niques are still developing quickly and we can
51
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